PET imaging of [52 Mn]Mn-DOTATATE and [52 Mn]Mn-DOTA-JR11

Manganese-52 is gaining interest as an isotope for PET imaging due to its desirable decay and chemical properties for radiopharmaceutical development. Somatostatin receptor 2 (SSTR2) is significantly overexpressed by neuroendocrine tumors (NETs) and is an important target for nuclear imaging and therapy. As an agonist, [68Ga]Ga-DOTATATE has demonstrated significant internalization upon interaction with receptor ligands, whereas [68Ga]Ga-DOTA-JR11(as an antagonist) exhibits limited internalization but better pharmacokinetics and increased tumor uptake. The goal of this study was to label both DOTATATE and DOTA-JR11 peptides with 52Mn in high radiochemical yields (RCY) and sufficient specific activity. A comparison of these two compounds was performed in in vitro and in vivo studies in animals with somatostatin receptor-positive xenografts to characterize differences in cell, tumor, and tissue uptake. Radiolabeling of DOTATATE and DOTA-JR11 was carried out by combining varying concentrations of the peptides with [52Mn]MnCl2. In vitro stability of the radiotracers was determined in mouse serum. In vitro cell uptake and internalization assays were performed in SSTR2 + AR42J cells and negative controls. In vivo biodistribution and longitudinal PET imaging was evaluated in mice bearing AR42J tumors. Both [52Mn]Mn-DOTATATE and [52Mn]Mn-DOTA-JR11showed affinity for SSTR2 in AR42J cells. However, the uptake of [52Mn]Mn-DOTATATE was higher (11.95 ± 0.71%/ mg) compared to [52Mn]Mn-DOTA-JR11 (7.31 ± 0.38%/ mg) after 2 h incubation. After 4 h incubation, 53.13 ± 1.83% of the total activity of [52Mn]Mn-DOTATATE was internalized, whereas only 20.85 ± 0.59% of the total activity of [52Mn]Mn-DOTA-JR11 was internalized. The PET images revealed similar biodistribution results, with [52Mn]Mn-DOTATATE showing a significant tumor uptake of 11.16 ± 2.97% ID/g, while [52Mn]Mn-DOTA-JR11 exhibited a lower tumor uptake of 2.11 ± 0.30% ID/g 4 h post-injection. The synthesis of both radiotracers was accomplished with high RCY and purity. The cell uptake and internalization of [52Mn]Mn-DOTATATE showed higher levels compared to [52Mn]Mn-DOTA-JR11. PET images of the radiotracers in AR42J tumor bearing mice demonstrated similar biodistribution in all organs except the tumor, with [52Mn]Mn-DOTATATE showing higher tumor uptake compared to [52Mn]Mn-DOTA-JR11. The variations in properties of these tracers could be used to guide further imaging and treatment studies.


Introduction
Radiolabeled peptides with positron-emitting radiometals including 68 Ga, 86 Y, and 64 Cu have been used in targeted Positron Emission Tomography (PET) imaging of SSTR2, a receptor highly expressed in neuroendocrine tumors (NETs) [1][2][3][4].Since the Food and Drug Administration (FDA), approved [ 68 Ga]Ga-DOTATATE in 2016, followed by [ 64 Cu]Cu-DOTATATE in 2020, SSTR2 PET/CT imaging has become standard of care for detecting, staging, and restaging NETs [1,5,6].Furthermore, SSTR2 imaging offers valuable insights into identifying patients who could potentially bene t from peptide receptor radionuclide therapy (PRRT) with SSTR2-targeted radiopharmaceuticals including [ 177 Lu]Lu-DOTATATE.DOTATATE, DOTATOC, and DOTANOC agents are all SSTR agonists where the mode of action involves internalization into tumor cells following the interaction between the ligand and the receptor [6,7].Conversely, SSTR antagonists do not undergo internalization into tumor cells upon receptor interaction, hence they were not initially seen as suitable targeting agents for the development of PET radiopharmaceuticals [4,5,[7][8][9][10].However, preclinical studies conducted by Ginji et al. among others, revealed that the radiolabeled SSTR antagonists can exhibit superior tumor-targeting capabilities compared to the agonists which is attributed to the ability of antagonists to identify and attach to a greater number of binding sites on the receptors [11].Similar ndings have been reported by various research groups that have investigated the impact of chelator, radiometal, and tumor type on tumor uptake [8,10,12].
Manganese-52, 52 Mn (t 1/2 = 5.6 d, β + = 29.6%,E βave = 242 keV) possesses favorable decay and chemical characteristics for PET imaging, especially in the development of long-lived PET radiopharmaceuticals.The 5.6-day half-life allows for enough time for the separation of the target, the process of radiochemistry, distribution to other sites, and PET imaging for several days following injection.The decay process includes a positron decay branching ratio of 29.6% and emits positrons with an average energy of 242 keV, which is comparable to that of 18 F (250 keV) [13][14][15][16][17].This results in high-resolution PET images.
Mn 2+ is a hard Lewis acid and exhibits a preference for coordination with hexadentate or heptadentate ligands containing N and O as donor atoms [13].The chelator DOTA has been successfully radiolabeled with 52 Mn and has demonstrated stability both in vitro and in vivo [14].In this current study, we aimed to assess and compare the targeting ability of [ 52 Mn]Mn-DOTATATE (agonist) and [ 52 Mn]Mn-DOTA-JR11(antagonist) in both in vitro and in vivo using PET imaging.

Materials and methods
Natural chromium powder (5 N purity) was purchased from ESPI metals (Ashland, OR). 1 mL SPE tubes with frits were sourced from Millipore Sigma (Burlington, MA).AG1-X8 analytical grade 200-400 mesh chloride form resin was obtained from Bio-Rad (Hercules, CA).DOTATATE was obtained from Macrocyclics (Plano, TX) while DOTA-JR11 was obtained from CPC scienti c (San Jose, CA).Mouse serum was sourced from EMD Millipore Corporation (Temecula, CA).Silica-embedded iTLC-SG paper was obtained from Sorbtech Technologies (Norcross, GA).A549, MCF7, and AR42J cells were all purchased from the American Type Culture Collection (Manassas, VA).All other chemicals and solvents were purchased from Fisher Scienti c (Hampton, NH) unless otherwise speci ed.

Production and puri cation of 52 Mn
52 Mn was produced by irradiation of natural chromium targets on a TR24 cyclotron (Advanced Cyclotron Systems Inc.) through the nat Cr(p,n) 52 Mn nuclear reaction, as described in previous studies [17,18].For puri cation, AGI-X8 resin packed in 1 mL solid-phase extraction (SPE) tubes was used, and the elution of [ 52 Mn]MnCl 2 was carried out in 6 M HCl as previously reported by Pyles et al. [17].
2.2 Radiolabeling of DOTATATE and DOTA-JR11 with 52 Mn DOTATATE and DOTA-JR11 were dissolved in water to make stock solutions at a concentration of 1 mg/mL.3.7 MBq (100 µCi) of ammonium acetate buffered [ 52 Mn]MnCl 2 (pH 4.5) was added to a 1.5 mL microcentrifuge tube containing 100 µL of 0.25 M ammonium acetate, pH 4.5, and varying concentrations of the ligands (0.1-1.0 nM).The reaction mixtures were incubated at 90°C while shaking for 30 min on a thermomixer (800 RPM).The radiochemical yields (RCY) of the radioligands were assessed using radio-TLC (instant thin layer chromatography) by using silica-embedded iTLC-SG paper.

Stability of the radioligands in mouse serum
For serum stability evaluation, 50 µL each of [ 52 Mn]Mn-DOTATATE and [ 52 Mn]Mn-DOTA-JR11 were separately mixed with 500 µL of mouse serum in triplicate and incubated at 37°C.At the speci ed time points, 40 µL of serum/radioligand mixture was collected and combined with an equal amount of methanol to separate serum proteins, as described in a previously published protocol on metabolite extraction and protein removal [19].The supernatant was analyzed using both radio TLC and radio HPLC, as described above to determine the percentage intact complex.
2.4 Cell studies 2.4.1 Cell culture A549 (human lung cancer) and MCF-7 (human breast cancer) cell lines were grown in Gibbco's high glucose Dulbecco' Modi ed Eagle's Medium (DMEM) that was supplemented with 10% fetal bovine serum (FBS) and 80 µM gentamicin.AR42J (rat pancreatic carcinoma) cell line was cultured in Roswell Park Memorial Institute (RPMI) 1640 media containing 20% FBS and 1 µg/mL gentamicin.The cells were maintained and grown in humidi ed incubators at 37°C with 5% CO 2 atmosphere.

Flow cytometry
Flow cytometry experiments were conducted to determine levels of SSTR2 expression.For uorescenceassociated cell sorting (FACS), MCF-7 and A549 cells were isolated from culture using 0.25% Trypsin and cell viability was assessed via Trypan blue exclusion method.Samples (n = 3 per cell line) were resuspended at 1x10 6 cells per mL of 1X FACS buffer (1% bovine serum albumin in phosphate-buffered saline), and subsequently incubated with uorophore-conjugated antibodies for 30 min in the dark at 4 ºC.Antibodies utilized were 1 µg SSTR2 (Novus, #MAB4224) conjugated with Cy5.5 (LumiProbe, 7321-10rxn), and Near-IR Dead Cell Stain Kit (ThermoFisher, L34975).Data was acquired on an Attune NxT ow cytometer obtained from ThermoFisher Scienti c (Waltham, MA, USA) and subsequent analysis was performed in FlowJo 10.6.2 software.Analysis of relative SSTR2 expression was determined using mean uorescent intensity (MFI) values, frequency, and count of SSTR2 + populations.

Cell uptake
1x10 5 AR42J, A549, and MCF-7 cells were seeded onto 24-well plates (n = 6) and incubated at 37°C 48 h before study.The incubating cell media was removed followed by addition of 1 mL of fresh media containing 0.5 nM of either [ 52 Mn]Mn-DOTATATE or [ 52 Mn]Mn-DOTA-JR11 with a speci c activity of ~ 0.68 ± 0.05 MBq/nmol (18.31 ± 1.05 µCi/nmol) into each well.The cells were incubated at 37°C under 5% CO 2 for 2 h.Following incubation, the media was removed, and the cells were washed 3 times with icecold PBS.500 µL of 0.2 M NaOH was added to detach the cells before collection into microcentrifugetubes followed by a 500 µL wash of PBS.Associated radioactivity was counted on a HIDEX (Turku, Finland) AGM gamma counter.
To normalize the counts to the total protein amount, a BCA protein assay (ThermoFisher Scienti c) was performed, and the nal data reported in % cell uptake/mg of protein.

Cell internalization
Following a published procedure with slight modi cations [20], 5× 10 5 AR42J and MCF7 cells were seeded to 12-well plates and incubated overnight at 37°C under 5% CO 2 .After removing the culture media, 1 mL of fresh media containing 0.5 nM of either [ 52 Mn]Mn-DOTATATE or [ 52 Mn]Mn-DOTA-JR11 with speci c activity of 0.37 MBq/µg was added to each well and the plates were incubated at 37°C under 5% CO 2 for various time points (30 min, 1, 2, 4, and 24 h).At each time point, the media was removed, and cells were washed 3 times with ice-cold PBS.
To determine the surface bound fraction, 0.5 mL of 0.1 M citric acid (pH 5) was added to each well and incubated for 5 min.The fraction was collected into a microcentrifuge tube followed by addition of 0.5 mL gentle wash of PBS.To determine the internalized activity, 0.5 mL of 0.2 M NaOH was added to each well and incubated at 37°C for 5 min.This fraction was collected in a separate tube followed by addition of 0.5 mL of PBS wash.The nal results were expressed as a percentage of the total activity that was present in each of the two fractions.

Animal studies
All animal studies were conducted using a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham and were compliant with national animal welfare policies and guidelines.
Female athymic nude mice purchased form Charles River lab (Charles River, Wilmington, MA) were left for one week to acclimate before initiating experiments.

Tumor implantation
Subcutaneous tumor xenograft models for AR42J were established by implanting 1x10 6 cells in complete cell media subcutaneously into the right shoulder 3-4 weeks (tumor average diameter of between 5-7 mm) before study.

PET/CT imaging and biodistribution
Approximately 2.22 ± 0.20 MBq (60.0 ± 5.4 µCi) of [ 52 Mn]Mn-DOTATATE or [ 52 Mn]Mn-DOTA-JR11 (6 µg) in 100 µL injection doses in saline were prepared.Mice (n = 4 per group) were anesthetized with 2.5% iso urane in oxygen and were injected via the retroorbital sinus.After injection, at 4, 24, and 48 h, mice were imaged on a So e GNEXT PET/CT small animal scanner (So e Biosciences, Dulles, VA, USA).At each time point, 30 min of PET data were acquired followed by a 3-min CT at 80 kVp for anatomical reference.At each time point post imaging, mice were euthanized, and organs were collected, weighed, and counted for associated radioactivity on gamma counter.Radioactivity uptake was calculated as the percent injected dose per gram of tissue (% ID/g).
For data processing, PET images were reconstructed via 3D-OSEM (Ordered Subset Expectation Maximization) algorithm (24 subsets and 3 iterations), with random, attenuation, and decay correction, and CT was reconstructed with the Modi ed Feldkamp Algorithm and analyzed using VivoQuant 4.0 (Invicro Imaging Service and Software, Boston MA) software.Following reconstruction of the images, regions of interest (ROIs) were hand-drawn for select organs using CT images to determine the standard uptake values (SUVs) using VivoQuant imaging software.

Statistical analysis
Data were expressed as mean ± SD.Comparisons were made using GraphPad Prism 9 purchased from GraphPad Software, LLC (Boston, MA, USA) running both ordinary one-way ANOVA and 2-way ANOVA.P values of less than 0.05 were considered signi cantly different.
The radiolabeling results were con rmed using both the iTLC (free 52 Mn moves with the solvent front with an R f =1 while radiolabeled peptides showed an R f =0.36) and HPLC (radiolabeled peptides elute at 10.5 min while free 52 Mn elute at 2 min) techniques as shown by the chromatograms in Fig. 1.

Serum stability
Serum stability tests of [ 52 Mn]Mn-DOTATATE and [ 52 Mn]Mn-DOTA-JR11 did not show signi cant decomplexation 5 days post incubation with mouse serum at 37°C with > 95% intact as assessed by radio-TLC (Fig. 2A).Only less than 10% of activity was associated with the precipitated proteins.HPLC chromatograms of mouse serum stability is provided in Fig. 2B.

Discussion
Various somatostatin analogs have been labeled with different PET radionuclides including 68 Ga, 18 F, and 64 Cu to aid in diagnostics, staging, and monitoring treatment outcomes [21]. 52Mn has gained signi cant interest for its positive PET imaging capabilities, including its su cient positron energy and extended half-life (5.6 d).The extended half-life enables imaging at later time points and facilitates distribution of the radionuclide after production [17].For this study, we explored the impact of using 52 Mn as the radionuclide to compare the in vitro and in vivo effectiveness of the agonist [ 52 Mn]Mn-DOTATATE and antagonist [ 52 Mn]Mn-DOTA-JR11 in mice bearing AR42J tumors.

Figure 2 A
Figure 2

Table 2 ,
[12]korah et al. have reported diverse observations regarding cellular cell uptake, but have obtained comparable results in terms of internalization with both the agonist and antagonist.During their comparative study on QGP1.SSTR2 cells, they examined the membrane binding and internalization of [ 18 F]AlF-NOTA-JR11 and [ 18 F]AlF-NOTA-TATE.After 1 h of incubation, they observed that [ 18 F]AlF-NOTA-JR11 had a binding rate of 85.2 ± 0.9%, with 5.1 ± 0.6% being internalized.In comparison, [ 18 F]AlF-NOTA-TATE had a binding rate of 34.9 ± 5.6%, with 23.5 ± 3.6% being internalized.These results deviate from the values we reported in this study, and the variation can be attributed to various factors such as structural changes, modi cations in radioelements, and variances in the incubation period.After 240 4h) indicate that the pharmacokinetics of both radioligands are comparable, except for the tumor and kidneys.The tumor uptake of [ 52 Mn]Mn-DOTATATE was much higher ( 11.16 ± 2.97% ID/g) Cu-CB-TE2A-Y3-TATE (2.86 ± 0.52% ID/g) 4 h after injection[12].In a separate study, Xie et al. found that the tumor uptake of [ 18 F]AlF-NOTA-JR11 was signi cantly lower (9.02 ± 5.9% ID/g) compared to [ 68 Ga]Ga-DOTATATE (31.35 ± 5.90% ID/g) in mice with HEK293-SSTR2 tumors[26].